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Bone Marrow Failure

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells

Mayumi Kamata,1 Yoko Okitsu,1 Tohru Fujiwara,1,2 Masahiko Kanehira,1 Shinji Nakajima,1 Taro Takahashi,1 Ai Inoue,1 Noriko Fukuhara,1 Yasushi Onishi,1 Kenichi Ishizawa,1 Ritsuko Shimizu,3 Masayuki Yamamoto,4 and Hideo Harigae1,2 Departments of Hematology and Rheumatology; 2Molecular Hematology/Oncology; 3Molecular Hematology; and 4Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan

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ABSTRACT

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of bone marrow mesenchymal stem cell differentiation is poorly understood. The transcription factor GATA2 is indispensable for hematopoietic stem cell function as well as other hematopoietic lineages, suggesting that it may maintain bone marrow mesenchymal stem cells in an immature state and also contribute to their differentiation. To explore this possibility, we established bone marrow mesenchymal stem cells from GATA2 conditional knockout mice. Differentiation of GATA2-deficient bone marrow mesenchymal stem cells into adipocytes induced accelerated oil-drop formation. Further, GATA2 loss- and gainof-function analyses based on human bone marrow mesenchymal stem cells confirmed that decreased and increased GATA2 expression accelerated and suppressed bone marrow mesenchymal stem cell differentiation to adipocytes, respectively. Microarray analysis of GATA2 knockdowned human bone marrow mesenchymal stem cells revealed that 90 and 189 genes were upregulated or downregulated by a factor of 2, respectively. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell cycle regulation, and the number of G1/G0 cells increased after GATA2 knockdown. Concomitantly, cell proliferation was decreased by GATA2 knockdown. When GATA2 knockdowned bone marrow mesenchymal stem cells as well as adipocytes were cocultured with CD34-positive cells, hematopoietic stem cell frequency and colony formation decreased. We confirmed the existence of pathological signals that decrease and increase hematopoietic cell and adipocyte numbers, respectively, characteristic of aplastic anemia, and that suppress GATA2 expression in hematopoietic stem cells and bone marrow mesenchymal stem cells.

Introduction Bone marrow mesenchymal stem cells (BM-MSC) are selfrenewing precursor cells that differentiate into bone, fat, cartilage, and stromal cells of the bone marrow, thereby forming a microenvironment that maintains hematopoietic stem cells.1 Accumulating evidence indicates the importance of the bone marrow microenvironment during hematopoietic cell development. Increased adipogenesis in the bone marrow negatively affects hematopoietic activity,2,3 whereas the osteoblastic niche supports hematopoietic stem cell function by activating Notch signaling.4 Therefore, precise regulation of BM-MSC differentiation into various lineages maintains hematopoiesis. Preadipocytes derived from MSC mature into adipocytes through a complex process involving numerous extracellular factors as well as transcription factors.1,5 Studies conducted on preadipocyte cell lines, such as mouse 3T3-L1 and 3T3-F442A, have uncovered the CCAAT/enhancer binding protein (C/EBP) family of transcription factors and the peroxisome proliferator-activated receptor γ (PPARγ) as key proadipogenic regulators.6,7 During preadipocyte–adipocyte differentiation, the expression of C/EBPβ and C/EBPδ initially increases, which subsequently activates the expression of C/EBPα and PPARγ, leading to the induction of genes involved in adipocyte function.8,9 However, the mechanism of differentiation of BMMSC into adipogenic progenitors and ultimately into mature

adipocytes in the bone marrow remains to be elucidated. GATA2, a transcription factor critically required in the genesis and/or function of hematopoietic stem cells (HSC),10-13 is expressed in various hematopoietic and non-hematopoietic tissues, including HSC, multipotent hematopoietic progenitors, erythroid precursors, megakaryocytes, eosinophils, mast cells, endothelial cells, and specific neurons.11,12,14-16 GATA2 is expressed by preadipocytes and BM-MSC and plays a central role in the control of adipogenesis.13,16,17 GATA2 overexpression in a mouse preadipocytic stromal cell line induces resistance to adipocyte differentiation, whereas GATA2 knockdown accelerates adipocyte differentiation,17 implying that GATA2 functions to arrest preadipocyte differentiation. Although GATA2 may suppress transcription of C/EBP and PPARγ in preadipocytes,16,18 the molecular mechanism by which GATA2 controls adipocyte differentiation remains unclear. Aplastic anemia is characterized by decreased HSC and fatty marrow replacement. Moreover, GATA2 expression is decreased in CD34-positive cells in aplastic anemia,19,20 Because BM-MSC express GATA2, it is possible that the signal that downregulates GATA2 expression in HSC may also suppress its expression in BM-MSC in aplastic anemia, thereby resulting in fewer HSC and an impaired microenvironment, which could support hematopoiesis. To test this hypothesis, we assessed the role of GATA2 during differentiation from BM-MSC.

©2014 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol.2014.105692 The online version of this article has a Supplementary Appendix. Manuscript received on February 12, 2014. Manuscript accepted on August 7, 2014. Correspondence: [email protected] 1686

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Regulation of BM-MSC differentiation by GATA2

Methods Generation of bone marrow mesenchymal stem cells To generate mouse BM-MSC, bone marrow cells from GATA2 conditional knockout mice were cultured in MesenCult MSC Basal Medium supplemented with 20% MSC stimulatory supplements (Stem Cell Technologies). The BM-MSC were transfected with the retroviruses expressing iCre to delete the DNA binding domain of GATA2 by inducing the Cre-loxP system.21,22 To generate human BM-MSC, bone marrow mononuclear cells from healthy donors were cultured with Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 20% fetal bovine serum (Life Technologies), 10 ng/mL basic fibroblast growth factor (PeproTech), 10 mM HEPES (Life Technologies), and 100 μg/mL penicillin/streptomycin (Invitrogen).23-25 Established BMMSC were used until the seventh generation. The study was approved by the ethical committee of Tohoku University Graduate School of Medicine. Clinical samples were collected after obtaining written informed consent. The ethics policies of the Declaration of Helsinki were followed.

Colony-forming cell assay CD34 positive-enriched cells, co-cultured with BM-MSC for 7 days, were seeded into semisolid culture (MethoCult™ H4435, Stem Cell Technologies). After 14 days, colony-forming units were counted.

Cell proliferation and cell cycle analysis The total number of viable cells was determined by a colorimetric method using MTS (3-4,5-dimethylthiazol-2-yl-5-3-carboxymethoxyphenyl-2-4-sulfophenyl-2H-tetrazolium, inner salt; CellTiter 96). Absorbance at 490 nm was measured with an iMark microplate reader (Bio-rad). For cell cycle analysis, cells were fixed in ice-cold 70% ethanol and stained with 20 mg/mL propidium iodide (Sigma), 0.2 mg/mL RNase (Sigma), and 0.1% Triton X-100 (Sigma). DNA content was determined using FACSAria II and FlowJo software (http://www.flowjo.com/).

Statistical analysis Statistical significance was assessed using a two-sided Student ttest.

Characterization of bone marrow mesenchymal stem cells BM-MSC immunophenotypes were determined using a FACSAria II (BD). To induce differentiation into adipocytes, human Mesenchymal Stem Cell Adipogenic Differentiation Medium (Lonza) was used. After 12–16 days, morphological changes were assessed using an inverted microscope. Typical adipocytes were stained with Oil Red O.2 The area of mature adipocytes was determined using HistoQuest software (Novel Science).

Quantitative reverse transcriptase polymerase chain reaction analysis and transcription profiling Quantitative reverse transcriptase polymerase chain reaction analysis (RT-PCR) was performed as previously described.26 Primer sequences are available upon request. For transcription profiling, the Human Genome U133 Plus 2.0 Array was used (Affymetrix). Gene ontology analysis was conducted using the DAVID bioinformatics program (http://david.abcc.ncifcrf.gov/).

Short interfering RNA-mediated knockdown Anti-GATA2 and control short interfering RNA (siRNA)26 were transfected into human BM-MSC with LipofectamineTM RNAiMAX reagent (Life Technologies). Cells were analyzed 48 h after transfection.

Viral vectors and cell transduction Retroviral overexpression of GATA2 was performed using the MSCV retrovirus vector, which co-expresses green fluorescent protein (GFP) by internal ribosome entry sites (IRES), transfecting into Platinum Retroviral Packaging Cell Lines (PLAT-F)27 with FuGENE HD (Roche). Human BM-MSC were pretreated with Retronectin (TAKARA BIO.), and GFP-positive cells were sorted using FACSAria II (BD Biosciences).

Co-culture of CD34-positive-enriched cells with a mesenchymal stem cell feeder layer BM-MSC were transfected with control or GATA2-siRNA. On day 3, control and GATA2 knockdowned BM-MSC, respectively, were replaced with serum-free medium containing CD34-positiveenriched cells (RIKEN). Serum-free medium (StemPro-34 SFM: Life Technologies) contained 100 ng/mL stem cell factor, 100 ng/mL interleukin (IL)-3, and 25 ng/mL granulocyte-monocyte colony-stimulating factor (Peprotech). The cells were co-cultured for 7 days, and subsequently harvested and analyzed with FACSAria II (BD). haematologica | 2014; 99(11)

Results Acceleration of adipocyte differentiation in mesenchymal stem cells from GATA2 knockout mice We first generated BM-MSC from bone marrow cells of conditional GATA2 knockout mice (GATA2fl), in which the DNA binding domain of GATA2 (exon 5 encoding the C-terminal zinc-finger motif) could be deleted by inducing the Cre-loxP system (GATA2−) (Online Supplementary Figure S1A). We confirmed that GATA2fl BM-MSC retained the potential to differentiate into adipogenic lineages (Online Supplementary Figure S1B). Flow cytometric analysis confirmed the characteristic immunophenotype,28 showing that GATA2fl BM-MSC expressed CD29, CD44 and Sca-1 but not markers such as CD11b, CD34 and CD45 (Online Supplementary Figure S1C). To determine whether the loss of GATA2 influenced the BM-MSC phenotype, the DNA-binding domain of GATA2 was deleted using the Cre-loxP system, and GATA2 knockout–BM-MSC (GATA2− BM-MSC) were generated. Quantitative RT-PCR analysis revealed that Gata2 expression was significantly decreased in the GATA2- MSC, implying that iCre-mediated deletion of the GATA2 C-finger resulted in decreased GATA2 autoregulation (Figure 1A). When GATA2fl and GATA2− BM-MSC were exposed to adipogenic differentiation stimuli, we observed an overall increase in the expression of Cebpa (CEBPα), Pparg (PPARγ), and Fabp4 (aP2) in GATA2− BM-MSC (Figure 1B). Moreover, the expression of these genes peaked during days 8–12 of differentiation and then dropped to levels similar to those of control cells (Figure 1B), whereas the expression level of Cebpb (CEBPβ) was slightly higher in GATA2− BM-MSC at the early (day 4) and last (day 16) stages of differentiation (Figure 1B). Furthermore, oil drop formation was markedly increased in GATA2– MSC (Figure 1C). These results suggest that loss of GATA2 function induces the expression of adipogenic factors and adipocyte differentiation of BM-MSC.

Generation and characterization of human bone marrow mesenchymal stem cells Next, to elucidate the role of GATA2 in the context of 1687

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human BM-MSC differentiation, we generated BM-MSC from human mononuclear cells derived from bone marrow samples. We confirmed that BM-MSC differentiated into the adipogenic lineage (Online Supplementary Figure S2A). Flow cytometric analysis further confirmed the characteristic immunophenotype,24,29,30 showing that the BM-MSC expressed CD29, CD44, CD90 and CD105 but not CD14, CD34, and CD45 (Online Supplementary Figure S2B).

Short interfering RNA-mediated GATA2 knockdown promotes differentiation of human bone marrow mesenchymal stem cells into adipocytes To determine whether GATA2 regulates adipocyte differentiation in human BM-MSC, we suppressed GATA2

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expression using a specific siRNA. Control or GATA2siRNA were transfected into human BM-MSC 48 h before inducing adipocyte differentiation. We demonstrated that GATA2 mRNA levels were significantly decreased on day 0 and during adipocyte differentiation until day 8 (Figure 2AB). Thereafter, we analyzed the expression of key adipocyte-specific genes at various time-points during adipocyte differentiation. The levels of expression of C/EBPα, PPARγ and aP2 were significantly increased in the GATA2-knockdowned cells (Figure 2B). Furthermore, oil drop formation on day 12 was significantly increased in the GATA2 knockdown cells, as determined based on the Oil Red O staining-positive area (Figure 2C-D). These findings were consistent with the results for GATA2-deficient murine BM-MSC (Figure 1).

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Figure 1. Effect of GATA2 depletion on the differentiation of murine MSC into adipocytes. GATA2-depleted BM-MSC (GATA2−) were generated by inducing iCre into the BM-MSC, established from GATA2 conditional knockout mice (GATA2fl). Each BM-MSC was then differentiated into the adipocyte lineage for up to 16 days. The data are representative of two independent BM-MSC lines. (A, B) Quantitative RT-PCR analysis for Gata2 expression on day 0 (A) and adipocytespecific genes during adipocyte differentiation (B). The expression of each target gene relative to that of Gapdh was calculated. The data are expressed as mean ± standard deviation, SD (n = 3). *P < 0.05. (C) Oil Red O staining on day 16. The picture is a representative example of at least three independent experiments. haematologica | 2014; 99(11)

Regulation of BM-MSC differentiation by GATA2

GATA2 overexpression suppresses differentiation of human bone marrow mesenchymal stem cells into adipocytes

Enrichment of cell cycle regulatory genes based on transcriptional profiling to identify GATA2-regulated genes in human bone marrow mesenchymal stem cells

We overexpressed GATA2 in human BM-MSC using MSCV-GFP-IRES. After transfecting GATA2-expressing or control retroviruses, GFP-positive cells were sorted. Quantitative RT-PCR assay confirmed GATA2 overexpression (Figure 3A, B). When these cells were differentiated into adipocytes, the levels of expression of C/EBPα, PPARγ, aP2 and Adipsin were significantly diminished by GATA2 overexpression (Figure 3B). Concomitantly, oil drop formation on day 12 was also significantly decreased in cells overexpressing GATA2 (Figure 3C,D). Taken together, our data suggest that decreased GATA2 expression by human BM-MSC accelerates adipocyte differentiation, whereas GATA2 overexpression suppresses adipocyte differentiation.

To identify GATA2-target genes in BM-MSC, we conducted comprehensive expression profiling of BM-MSC transfected with control or GATA2-siRNA. Inhibition of GATA2 expression was confirmed based on the profiling data as well as quantitative RT-PCR analysis (0.000104 ± 0.000008 and 0.000198 ± 0.000022, for GATA2 siRNA and control siRNA, respectively, P 2-fold), respectively (Table 1, Online Supplementary Table S1). The analysis revealed the differential expression of cell-cycle regulators (CHEK1, CCNB1, CCNB2, GTSE1, and CDC20), adhesion molecules (LAMP1 and CD44), as well as

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Figure 2. Effects of GATA2 knockdown on the differentiation of human MSC into adipocytes. GATA2 expression was suppressed in human BMMSC by transfecting them with a human GATA2siRNA, and the cells were induced to differentiate into the adipocyte lineage for up to 12 days. The data are representative of three independent BMB) MSC lines. (A, Quantitative RT-PCR analysis for GATA2 expression on day 0 (A) and adipocyte-specific genes during adipocyte differentiation (B). The expression of each target gene relative to that of GAPDH was calculated. (C, D) Oil Red O staining (C) and the area of mature adipocytes (D). The data are expressed as mean ± SD (n=3). *P

GATA2 regulates differentiation of bone marrow-derived mesenchymal stem cells.

The bone marrow microenvironment comprises multiple cell niches derived from bone marrow mesenchymal stem cells. However, the molecular mechanism of b...
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